1. Field of the Invention
The present invention relates to a culturing method of mesenchymal stem cells which can select and proliferate the mesenchymal stem cells efficiently without necessity of a specialized separating device and a complicated separating operation.
2. Description of the Conventional Art
Since the mesenchymal stem cells included in a bone marrow solution, an umbilical cord blood, a peripheral blood or the like has a multi-differentiation potency potent capable of differentiating into many kinds of cells such as those of a bone, a cartilage, a fat and the like, attention has been attracted to the mesenchymal cells as a cell source for a cell therapy and a regeneration medical treatment (refer, for example, to International Publication WO01/83709). However, the mesenchymal stem cell has a very low existence frequency, for example, only one per 104 to 106 cells existing in the bone marrow solution of an adult, and in the case of use for a clinical treatment, it is necessary to culture and proliferate the mesenchymal stem cells collected from tissue so as to use them.
In order to use the mesenchymal stem cells as the cell source for the regeneration medical treatment, it is important to secure the mesenchymal stem cells by a method having a high efficiency particularly at a time of a primary culture (a culture until a first replanting is carried out after taking out of a living body), and move to a successive culture (a culture for proliferating and maintaining the cultured cells by removing the cultured cells to a new culture vessel). For example, it is necessary to implant a fixed number or more mesenchymal stem cells for obtaining a good medical treatment result, and it is advantageous if an efficiency of the cell culture from the same amount of a bone marrow solution, an umbilical cord blood, a peripheral blood or the like as their source is high, and the number of the cells obtained by the primary culture is large.
It is well known that in the course of proliferation, senile change of the cells appears uniformly. It has been also known that if the senile change makes progress, a differentiation potency of the cells to a tissue is lowered. If the differentiation potency of the implanted cells is low, a tissue regeneration performance is lowered, and a medical treatment effect is lowered. If the number of the cells obtained at an initial stage is small, additional proliferation is necessary. Accordingly, the senile change makes progress additionally at a time of reaching the fixed number of cells, and a risk is caused.
Further, it has been known that a proliferating capacity is lowered by the senile change. In case that obtained cell number is small, the additional proliferation is necessary. So, the culturing period becomes extremely elongated due to a reduction of the proliferating capacity. In order to establish the medical treatment on the basis of the cell implanting in the future, it is necessary to take a cost thereof into consideration. If the culturing period becomes longer, the cost for the medical treatment becomes higher.
Further, if the efficiency of the primary culture is improved, it is not only possible to reduce an amount of the bone marrow gathered from a patient, to thereby reduce an infestation and a burden applied to the patient, but also possible to freeze and reserve the cells which are surplus after the medical treatment, so as to prepare for a future disease because a lot of cells can be obtained.
The mesenchymal stem cell is a spindle shaped adhesive cell like a fibroblast, and in order to culture the mesenchymal stem cells corresponding to the adhesive cells, it is first of all necessary to adhere to a bottom surface of a plastic dish for culturing. Thereafter, the mesenchymal stem cells are separated from a body fluid by removing non-adhered components such as blood cells or the like mixing in a sample. At this time, since the blood cells such as a red blood cells, a neutrophil, a lymphocyte, a basopil, an eosinophil or the like have larger specific gravities than a specific gravity of the mesenchymal stem cells and settle down earlier, and the number of the blood cells is larger than that of the mesenchymal stem cells in a culture solution, most of the bottom surface of the plastic dish for culturing is covered by those blood cells. As a result, a space for adhering which is necessary for the mesenchymal stem cells is necessarily reduced, and it becomes impossible to obtain a sufficient amount by the primary culture.
Further, in the case that the sample gathered from the living body is constituted by a tissue such as a jaw bone marrow solution, an umbilical cord blood, a peripheral blood or the like, an existence rate of the blood cells or the like is larger in comparison with bone marrow solutions of an iliac bone, a femur or the like. Accordingly, if a seeding amount of the sample is increased for raising a culture density of the mesenchymal stem cells, a density of the blood cells such as the red blood cell or the like becomes higher in proportion thereto, and there is a problem that the adhesion of the mesenchymal stem cells is obstructed.
Then, a method of separating the mesenchymal stem cells from the blood cells such as the red blood cell or the like before culturing has been conventionally carried out. In accordance with this method, it is possible to prepare the mesenchymal stem cells existing in a mononuclear cell fraction at a high purity, however, since aiming at and extracting a fraction having a specific density from a centrifugally separated layer not only require a specialized device and a complicated operation, but also demand a knowledge and a skilled technique, it has been hard to carry out this method commercially. Further, it is necessary to carry out a washing operation again and again for picking up the mesenchymal stem cells from the centrifugally separated layer, the mesenchymal stem cells may be washed away during this process, and it is impossible to efficiently separate the mesenchymal stem cells.
Further, a method of separating and collecting the stem cells by using a filter-shaped stem cell separating device having specific density and fiber diameter is disclosed (refer, for example, to International Publication WO02/46501). This method is a method capable of separating and collecting the stem cells from the bone marrow solution without necessity of addition of a separating reagent and necessity of a centrifugal separating operation. However, since the stem cells are left in the separation material, the method has a great loss and is not efficient.
Further, there exists a separating method of targeting a surface antigen which is characteristic for the mesenchymal stem cell (refer, for example, to Ishimura, D., et al., Differentiation of adipose-derived stromal vascular fraction culture cells into chondrocytes using the method of cell sorting with a mesenchymal stem cell marker. Tohoku J Exp Med, 2008. 216(2): p. 149-56.). It is possible to mark the mesenchymal stem cell corresponding to a target cell by adding a micro bead having such a magnetism as to combine with an antibody against the surface antigen to the sample such as the gathered bone marrow or the like. If the sample is adapted to a separation column put in a magnetic field, the mesenchymal stem cells marked by the magnetism stay in the column, and the other unnecessary cells can be flowed out of the column, whereby it is possible to separate the target cells. The mesenchymal stem cells can be washed out of the column by removing the column from the magnetic field (refer, for example, to Japanese Unexamined Patent Publication No. 2004-254519). Since this method uses the antibody, this is a method capable of more selectively obtaining the mesenchymal stem cells. However, this method has a great loss in the thereafter washing step. Further, there is a risk of an effect by the mesenchymal stem cell joined with the antibody being applied as it is to a clinical treatment and so forth. Further, an exclusive device such as a special antibody, a magnetic bead, a system providing a magnetic field, a column or the like is necessary.